This project addresses 3D or virtual models to reduce the use of animals in research: Creation of miniature multi-cellular organs for high-throughput screening for chemical toxicity testing. Our long-term objective is to decrease the use of animals for toxicity assays and increase the use of human cells for toxicity and safety assessment. We will establish a three-dimensional (3D) model of the normal human skin to create experimental conditions that are more 'human-specific'and more predictive with the added benefit of decreasing the number of animals required for preclinical toxicology and safety assessment. Mouse skin is significantly different from human skin in its architecture and varies in response to environmental toxins (biological origin) and xenobiotics (synthetic chemicals). Our working hypothesis is that 3D reconstructed human skin can be used as a reliable system to screen for the effects of xenobiotic exposures. The murine epidermis is very thin and largely devoid of melanocytes, whereas human epidermis is thick and contains pigment-producing melanocytes. Major carcinogens for mouse skin such as DMBA and the phorbol ester PMA/TPA commonly used in classical two-stage carcinogenesis studies in mice show little effect on human skin grafted to immunodeficient animals. To better determine which xenobiotics are affecting human cells, we will reconstruct in vitro human skin containing two layers: 1) 'dermis'consisting of fibroblasts and, optionally, also endothelial, smooth muscle, and inflammatory, and immune cells that are all embedded in collagen type I;2) 'epidermis'consisting of multilayered keratinocytes at distinct differentiation stages and melanocytes, which reside in the basal layer and attach to the developing basement membrane. Synthetic human skin (also termed skin reconstructs or organotypic skin) contains at least three cell types (fibroblasts, melanocytes, keratinocytes) and up to six additional cell types (e.g., including endothelial cells, smooth muscle cells, and inflammatory cells), and the structures and functions generated are similar to intact, full-thickness human skin. Our specific objectives are: Specific Aim 1. Test human skin reconstructs of various complexities and develop parameters for detecting skin cell damage after exposure to xenobiotics. As parameters for measures of toxicity, we will use cell viability or death, changes in differentiation patterns for keratinocytes, melanocytes, and endothelial cells, and for long-term effects changes in markers associated with transformation. Specific Aim 2. Miniaturize reconstructs to 96-well format and establish standardized criteria for medium-to-high throughput testing. The human skin reconstructs will be established in 96-well plate format and we will perform side-by-side comparison of the in vitro models with the orthotropic human and the murine skin models to compare sensitivity, reproducibility, and interpretability of large-scale 3D testing of xenobiotics. Public Health Relevance Statement These studies aim to establish a three-dimensional (3D) model of the normal human skin to create experimental conditions that are more 'human-specific'and more predictive with the added benefit of decreasing the number of animals required for preclinical toxicology and safety assessment. We will perform side-by-side comparison of the in vitro models with the orthotropic human and the murine skin models to compare sensitivity, reproducibility, and interpretability of large-scale 3D testing of xenobiotics.